Anaerobic Sludge Digestion Liquor Research Articles

Advance nitrogen removal from anaerobic sludge digestion liquor using partial nitrification and denitrification coupled with simultaneous partial nitrification, anammox, and denitrification process

A novel two-stage continuous-flow partial nitrification and denitrification coupled with simultaneous partial nitrification, anammox, and denitrification (PND-SNAD) process was developed to treat anaerobic sludge digestion liquor. During the stable phase, the total nitrogen and chemical oxygen demand (COD) removal efficiencies were 93 ± 3 % and 59 ± 7 %, respectively. Free ammonia suppression (26.2 ± 12.7 mg/L) and low dissolved oxygen (DO, 0.12 ± 0.07 mg/L) were key factors in the operation of the PND process, while the SNAD process was restricted successfully by limited oxygen (DO < 0.1 mg/L) and short solids retention time (9.7 d). The PND process was an important pretreatment process that could remove biodegradable dissolved COD by denitrification and supply ammonium-oxidizing bacteria (AOB) to the SNAD process. Nitrosomonas and Ca. Brocadia were the dominant AOB and anammox bacteria, respectively. Overall, this research presents a distinctive SNAD combined process for anaerobic sludge digestion liquor treatment.

Free ammonia shock treatment eliminates nitrite-oxidizing bacterial activity for mainstream biofilm nitritation process

Nitritation (NH4+ → NO2−) is a critical step to provide nitrite for the followed anammox in a two-stage nitrogen removal system. In the mainstream line of wastewater treatment plants, the nitritation has not been applied to date, because of the significant difficulties in the suppression of nitrite-oxidizing bacteria (NOB), especially in biofilm systems. This study aims to systematically assess the effect of free ammonia (FA) shock treatment on the mainstream biofilm nitritation process through an integration of laboratory reactor operation, microbial community analysis, incubation tests, and kinetic model evaluation. In a laboratory nitrifying moving bed biofilm reactor (MBBR) fed with domestic-strength synthetic wastewater, it was shown that with the exposure of carrier-biofilms containing ammonia-oxidizing bacteria (AOB) Nitrosomonas and NOB Nitrospira to a high-level of FA (1068 mg NH3-N/L) over two days, a much higher residual AOB level was retained in the biofilm in comparison to NOB. The higher residual AOB on biofilm led to much faster recovery of AOB over NOB after the shock treatment, when normal operation resumed with the dissolved oxygen (DO) controlled at around 0.2 mg/L. The faster recovery of AOB than NOB subsequently gave rise to a stable, high nitrite accumulation ratio (nearly 100%) over a long period (two months). Collectively, these results suggest that FA shock treatment in conjunction with limited DO control is effective in eliminating NOB for mainstream biofilm nitritation process. The chemical cost would be marginal given the intermittent nature of the FA shock strategy and the readily available ammonium in the anaerobic sludge digestion liquor.

High-level nitrogen removal by simultaneous partial nitritation, anammox and nitrite/nitrate-dependent anaerobic methane oxidation

While the anaerobic ammonium oxidation (anammox) process has been applied for nitrogen removal from high-strength wastewater, nitrate accumulation in effluent still represents a major concern. Here, a novel process, named the one-stage PNAM, that integrates the Partial Nitritation (PN), Anammox and Methane-dependent nitrite/nitrate reduction reactions in a single membrane biofilm reactor (MBfR) is developed. With feeding of 1030 mg NH4+-N/L at a hydraulic retention time of 16 h, the proposed one-stage PNAM process achieved an average total nitrogen removal efficiency of 98% and a nitrogen removal rate of 1.5 kg N/m3/d (1.4–1.8 g N/m2/d) by using methane as the sole carbon-based electron donor. The N2O emission was determined to be 0.34% ± 0.01%. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) bacteria and archaea co-developed in the biofilm. Batch tests showed that AOB, anammox bacteria and n-DAMO microorganisms were indeed jointly responsible for the nitrogen removal. This one-stage PNAM process can potentially be applied to treating high-strength wastewater, such as anaerobic sludge digestion liquor or landfill leachate.

Simple technology for recycling phosphate from wastewater to farmland in rural areas

A simple technology for phosphate (Pi) recovery has been developed using a bifunctional adsorption–aggregation agent. The bifunctional agent was prepared by soaking calcium silicates in hydrochloric acid solution. Importantly, recyclable calcium silicates were available almost free of charge from the cement industry and also from the steel industry. The acid treatment was essential not only for enhancing the ability of calcium silicates to remove Pi from aqueous solution but also for enabling the high settleability of removed Pi. On-site experiments using a mobile plant showed that approximately 80% Pi could be recovered from anaerobic sludge digestion liquor at a wastewater treatment plant. This technology has the potential to offer a simple, compact service for recycling Pi from wastewater to farmland in rural areas.

A pilot-scale study on the start-up of partial nitrification-anammox process for anaerobic sludge digester liquor treatment

Treatment of sludge digester liquor was successfully accomplished using a pilot-scale partial nitrification-anammox (PN/A) reactor with a nitrogen removal rate (NRR) of 1.23kgN/m3/d. A stable and efficient PN process was attained by controlling the concentration of free ammonia (0.7–8.4mg/L) and free nitrous acid (0.02–1.0mg/L). The application of hydroxylamine played a vital role in the reactivation of anammox bacteria. The bacteria exhibited improved granule properties at a specific input power between 0.065 and 0.097kW/m3, and achieved a specific anammox activity (SAA) of 1.01kgN/kgVSS/d on day 148. From day 0 to 120, the heme c content in the granules increased from 0.42±0.1 to 5.77±1.0µmol/gVSS, with a corresponding increase in NRRs and SAAs. High-throughput sequencing techniques revealed that the dominant anammox bacterial genus was Candidatus Brocadia. These conclusions provide valuable information for the full-scale treatment of sludge digester liquor.

Complete Nitrogen Removal from Synthetic Anaerobic Sludge Digestion Liquor through Integrating Anammox and Denitrifying Anaerobic Methane Oxidation in a Membrane Biofilm Reactor.

Partial nitritation and Anammox processes are increasingly used for nitrogen removal from anaerobic sludge digestion liquor. However, their nitrogen removal efficiency is often limited due to the production of nitrate by the Anammox reaction and the sensitivity to the nitrite to ammonium ratio. This work develops and demonstrates an innovative process that achieves complete nitrogen removal from partially nitrified anaerobic sludge digestion liquor through the use of a membrane biofilm reactor (MBfR), with methane supplied through hollow fiber membranes. When steady state with a hydraulic retention time (HRT) of 1 day was reached, the process achieved complete nitrite and ammonium removal at rates of 560 mg N/L/d and 470 mg N/L/d, respectively, without any nitrate accumulation. The process is relatively insensitive to the nitrite to ammonium ratio, achieving complete nitrogen removal when their ratio in influent varied in the range of 1.125-1.32. Pyrosequencing and fluorescence in situ hybridization analysis revealed that denitrifying anaerobic methane oxidation (DAMO) archaea, Anammox bacteria and DAMO bacteria jointly dominated the microbial community. Mass balance analysis showed that nitrate produced by Anammox (122.2 mg N/L/d) was entirely converted to nitrite by DAMO archaea, while nitrite in the feed and produced by DAMO archaea was jointly removed by Anammox (90%) and DAMO bacteria (10%). The nitrogen removal rate of over 1 kg N/m3/d is comparable to the practical rates reported for side-stream nitrogen removal processes.

A mobile pilot-scale plant for in situ demonstration of phosphorus recovery from wastewater using amorphous calcium silicate hydrates

Abstract A mobile pilot-scale plant was developed for the in situ examination and demonstration of phosphate (P i ) recovery from wastewater. The mobile pilot plant consisted of a 1000-L reinforced-plastic reactor, a self-made filter, and ancillary equipment and was set up on a 1.5-tonne motor truck for transport. Five separate in situ experiments were carried out using the mobile pilot plant to evaluate the ability of amorphous calcium silicate hydrates (A-CSHs) to recover P i from anaerobic sludge digestion liquor at a wastewater treatment plant. On average, approximately 80% P i could be recovered from the anaerobic sludge digestion liquor by a process consisting of 20-min mixing, 30-min settling, and 90-min filtration. Approximately 20% of the dry weight of the recovered product was citrate-soluble P 2 O 5 . The levels of heavy metals such as Cd, As, Pb, Ni, and Cr were much lower than their regulatory standards for fertilizer. The efficacy of the recovered product as P i fertilizer was confirmed by plant cultivation tests using the leaf vegetable Komatsuna ( Brassica rapa L. var. perviridis ). The present study showed that the mobile pilot plant is useful as a simple, potentially low-cost tool for the in situ examination and demonstration of P i recovery from wastewater.

A simple technology for phosphorus recovery using acid-treated concrete sludge

Abstract A simple technology for phosphorus (P) recovery from aqueous solution has been developed using acid-treated concrete sludge (A-CS) as a bifunctional adsorption-aggregation agent. Dried particles of concrete sludge (CS), an alkaline waste containing hydrated cement and fine aggregates, were soaked in 1.3 M HCl at a concentration of 0.1 g/mL for 60 min. The HCl-soaking treatment solubilized alkaline substances such as Ca(OH) 2 and CaCO 3 , thereby releasing approximately 87% Ca 2+ from the CS particles to the acid solution. When A-CS was added to 500-mL synthetic anaerobic sludge digestion liquor containing 273 mg/L of phosphate (P i ) at the Ca/P molar ratio of 2.5, A-CS showed 20 times higher P i removal efficiency than that of untreated CS particles. Although A-CS could precipitate 72% P i in 5-min free sedimentation, the Ca 2+ -rich liquid fraction of A-CS alone led to the precipitation of only 48% P i . This suggests that the solid fraction of A-CS can serve as an auxiliary aggregation agent. When P i recovery was examined using anaerobic sludge digestion liquor from a full-scale wastewater treatment, A-CS could recover 96% P i at the Ca/P molar ratio of 2.5. Citrate-soluble P 2 O 5 accounted for 19% of the dry weight of the recovered P i product. The levels of heavy metals such as As, Cd, Pb, Ni, and Cr in the recovered P i product were much below their regulatory standards for fertilizers.

A new approach to simultaneous ammonium and dissolved methane removal from anaerobic digestion liquor: A model-based investigation of feasibility

The presence of a high level of dissolved methane (e.g., 20–26 g m−3) in the anaerobic sludge digestion liquor represents a major challenge to the treatment of this stream, as its emission to the atmosphere contributes significantly to the carbon footprint of wastewater treatment. Here we propose a new approach to simultaneous ammonium and dissolved methane removal from the anaerobic digestion liquor through integrating partial nitritation-Anammox and denitrifying anaerobic methane oxidation (DAMO) processes in a single-stage membrane biofilm reactor (MBfR). In such an MBfR, the anaerobic digestion liquor is provided in the bulk liquid, while oxygen is supplied through gas-permeable membranes to avoid dissolved methane stripping. A previously developed model with appropriate extensions was applied to assess the system performance under different operational conditions and the corresponding microbial interactions. Both influent surface loading (or hydraulic retention time) and oxygen surface loading are found to significantly influence the total nitrogen (TN) and dissolved methane removal, which jointly determine the overall system performance. The counter diffusion and concentration gradients of substrates cause microbial stratification in the biofilm, where ammonia-oxidizing bacteria (AOB) attach close to the membrane surface (biofilm base) where oxygen and ammonium are available, while Anammox and DAMO microorganisms jointly grow in the biofilm layer close to the bulk liquid where methane, ammonium, and nitrite are available with the latter produced by AOB. These results provide first insights and useful information for the design and operation of this new technology for simultaneous ammonium and dissolved methane removal in its potential future applications.

Amorphous calcium silicate hydrates and their possible mechanism for recovering phosphate from wastewater

Amorphous calcium silicate hydrates (A-CSHs) were synthesized using soluble silicates extracted from a natural siliceous shale (M-rite) and Ca(OH)2. Simultaneous thermogravimetry and differential thermal analysis confirmed that the synthesized A-CSHs contained no detectable amount of free Ca(OH)2. Their performance on phosphate (Pi) recovery from aqueous solutions was examined using a 3.0-L bacth reactor. A-CSHs possessed a greater ability to recover Pi from a synthetic anaerobic sludge digestion liquor than did CaCl2 and Ca(OH)2. 29Si magic-angle-spinning NMR analysis suggested that wet A-CSHs consisted of silicate polymers (average chain length of 3.5) that are linked to each other through ion binding with Ca2+. Based on Ca2+ release and settleability experiments, it was speculated that Ca–Pi–silicates aggregates were formed by the ionic association of Pi, Ca2+, and negatively charged silicates. This hypothesis could reasonably explain the high settleability of Pi removed by A-CSHs. Powder X-ray diffraction analysis showed that recovered products had an amorphous structure similar to that of A-CSHs. In this study, we suggest that A-CSHs have a unique mechanism for recovering Pi, thereby enabling their high reactivity and settleability.

N2O production by ammonia oxidizing bacteria in an enriched nitrifying sludge linearly depends on inorganic carbon concentration

The effect of inorganic carbon (IC) on nitrous oxide (N2O) production by ammonia oxidizing bacteria (AOB) was investigated over a concentration range of 0–12 mmol C/L, encompassing typical IC levels in a wastewater treatment reactors. The AOB culture was enriched along with nitrite-oxidizing bacteria (NOB) in a sequencing batch reactor (SBR) to perform complete nitrification. Batch experiments were conducted with continuous carbon dioxide (CO2) stripping or at controlled IC concentrations. The results revealed a linear relationship between N2O production rate (N2OR) and IC concentration (R2 = 0.97) within the IC range studied, suggesting a substantial effect of IC on N2O production by AOB. Similar results were also obtained with an AOB culture treating anaerobic sludge digestion liquor. The fundamental mechanism responsible for this dependency is unclear; however, in agreement with previous studies, it was observed that the ammonia oxidation rate (AOR) was also influenced by the IC concentration, which could be well described by the Monod kinetics. These resulted in an exponential relationship between N2OR and AOR, as previously observed in experiments where AOR was altered by varying dissolved oxygen and ammonia concentrations. It is therefore possible that IC indirectly affected N2OR by causing a change in AOR. The observation in this study indicates that alkalinity (mostly contributed by IC) could be a significant factor influencing N2O production and should be taken into consideration in estimating and mitigating N2O emissions in wastewater treatment systems.

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors.

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale "fill-and-draw" reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.

Producing free nitrous acid – A green and renewable biocidal agent – From anaerobic digester liquor

• Free nitrous acid (FNA) is produced from anaerobic digestion liquor (DL). • Complete nitritation of the DL is achieved with online pH controller. • Nitrite in the treated DL required minimal acid for its conversion to FNA. • Only 0.08% of N converted was emitted as nitrous oxide. Recent studies have shown that free nitrous acid (FNA) at parts per million is strongly biocidal to a broad range of microorganisms involved in wastewater management. Applications have been developed, where FNA is used to deactivate anaerobic sewer biofilms thus suppressing sulfide and methane production in sewers, or to lyse secondary sludge resulting in reduced sludge production and enhanced biogas production. This study examines the feasibility of producing FNA from a waste stream namely the anaerobic sludge digestion liquor, thus providing a source of FNA for the above applications within wastewater systems. Complete nitritation was achieved in a lab scale sequencing batch reactor (SBR) treating reject wastewater. Under stable operation, the system sustained more than 90% conversion of the 1.0 and 0.8 g NH 4 + -N/L contained in the synthetic and real digester liquor, respectively, to nitrite. Each liter of this nitrite rich effluent could be acidified to pH 2 with only 66 mmol of H + , due to the low level of alkalinity in the effluent. This converts almost all of the nitrite to FNA providing an ample source of FNA for sewer and sludge pretreatment applications. Despite the high nitrite concentration in the reactor, minimal N 2 O was produced with an emission factor of 0.08% of the ammonium nitrogen converted. Finally, an economical assessment of a theoretical full-scale installation for FNA production was conducted and compared with the costs of producing this FNA from a commercial nitrite supply.

Heterotrophic denitrification plays an important role in N2O production from nitritation reactors treating anaerobic sludge digestion liquor

Nitrous oxide (N2O) emissions from nitritation reactors receiving real anaerobic sludge digestion liquor have been reported to be substantially higher than those from reactors receiving synthetic digestion liquor. This study aims to identify the causes for the difference, and to develop strategies to reduce N2O emissions from reactors treating real digestion liquor. Two sequencing batch reactors (SBRs) performing nitritation, fed with real (SBR-R) and synthetic (SBR-S) digestion liquors, respectively, were employed. The N2O emission factors for SBR-R and SBR-S were determined to be 3.12% and 0.80% of the NH4+-N oxidized, respectively. Heterotrophic denitrification supported by the organic carbon present in the real digestion liquor was found to be the key contributor to the higher N2O emission from SBR-R. Heterotrophic nitrite reduction likely stopped at N2O (rather than N2), with a hypothesised cause being free nitrous acid inhibition. This implies that all nitrite reduced by heterotrophic bacteria was converted to and emitted as N2O. Increasing dissolved oxygen (DO) concentration from 0.5 to 1.0 mg/L, or above, decreased aerobic N2O production from 2.0% to 0.5% in SBR-R, whereas aerobic N2O production in SBR-S remained almost unchanged (at approximately 0.5%). We hypothesised that DO at 1 mg/L or above suppressed heterotrophic nitrite reduction thus reduced aerobic heterotrophic N2O production. We recommend that DO in a nitritation system receiving anaerobic sludge digestion liquor should be maintained at approximately 1 mg/L to minimise N2O emission.

Novel technique for phosphorus recovery from aqueous solutions using amorphous calcium silicate hydrates (A-CSHs)

A novel technique for phosphorus (P) recovery from aqueous solutions was developed using amorphous calcium silicate hydrates (A-CSHs). A-CSHs, which have a high Ca/Si molar ratio of 2.0 or greater, could be synthesized using unlimitedly available, inexpensive materials such as siliceous shale and calcium hydroxide. A-CSHs showed high performance for P recovery from an anaerobic sludge digestion liquor (ASDL) and the synthetic model liquor (s-ASDL) containing 89 mg PO4–P/L. After 20 min mixing, 1.5 g/L A-CSHs could remove approximately 69 and 73% PO4–P from ASDL and s-ASDL, respectively. By contrast, autoclaved lightweight concrete particles, which contained crystalline calcium silicate hydrates as a principal component, removed only 10 and 6% PO4–P from ASDL and s-ASDL, respectively, under the same experimental conditions. When A-CSHs were washed with deionized water to remove free Ca(OH)2, P removability was significantly improved (up to 82%) despite the reduction in the amount of Ca2+ released. Unlike in the case of Ca(OH)2, no significant carbonate inhibition was observed with P removal by A-CSHs. Moreover, P removed by A-CSHs showed better settleability, filterability, and dewaterability than P precipitated with conventional CaCl2 and Ca(OH)2. The present study demonstrated that A-CSHs have great potential as a novel, beneficial material for P recovery and recycling.

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1week at a nitrogen loading rate of 0.46 kg-N/m(3)/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m(3)/day at a nitrogen loading rate of 1.0 kg-N/m(3)/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m(3)/day at a nitrogen loading rate of 0.93 kg-N/m(3)/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5-6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.

Partial Nitritation of Raw Anaerobic Sludge Digester Liquor by Swim-Bed and Swim-Bed Activated Sludge Processes and Comparison of Their Sludge Characteristics

This study evaluated performance of swim-bed (SB) reactors packed with a novel acrylic fiber carrier (BF) and swim-bed activated sludge (SBAS) reactor for partial nitritation of anaerobic sludge digester liquor from a municipal wastewater treatment plant. Comparison of characteristics of sludge obtained from both the reactors was also made. The average conversion rates of ammonium to nitrite were 52.3% and 40.0% under relatively high nitrogen loading rates over 3.0 kg-N/m(3)/d, respectively in two reactors. The average BOD(5) removal efficiencies were 74.3% and 64.4%, respectively in the two reactors. The size of the sludge pellets taken from SB and SBAS reactors was found to be approximately three times (229 mum versus 88 mum) of that of the seed sludge. This sludge also had relatively high extracellular proteins levels indicating better sludge settling capability as compared to the sludge taken from SBAS reactor. Although the effluent nitrite/ammonium ratios had fluctuated in both reactor in some extent, the low dissolved oxygen concentration (average of 2.5 versus 0.35 mg/l), low suspended solids (average of 33.3 versus 33.5 mg/l), and about 50% ammonium conversion to nitrite demonstrated the application potential of anammox process for nitrogen removal.

Anammox process for synthetic and practical wastewater treatment using a novel kind of biomass carriers

The anammox process, as an alternative to conventional nitrogen removal technologies, has abstracted much attention in recent years. In this study, one column-type reactor using a novel support material--net type acrylic fiber (Biofix)--was used for anammox treatment. The Biofix reactor was operated at 25 degrees C (peak summer temperature, 31.5 degrees C). Over 330 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m3/d and T-N removal efficiencies reached to 81.3%. For the practical anaerobic sludge digester liquor treatment, the average TN removal efficiency of 72% was obtained. A protein substance was shown to be the most abundant extracellular polymeric substances (EPS) in the granular sludge with almost two times more in the attached sludge of the Biofix reactor. Considering the EPS levels and observation by scanning electron microscopy, the anammox granules in the Biofix reactor were showing dense state. Results of DNA analyses indicated that the KSU-1 strain might prefer relatively low nutrient levels, while the KU2 strain might be better suited for the high media concentration. Other kinds of bacteria were also identified with the potentials for consuming the dissolved oxygen in the influent and facilitating anammox bacteria surviving under aerobic conditions.

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25 degrees C (peak summer temperature, 31.5 degrees C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m(3)/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m(3)/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.